Many bioactive molecules form colloidal aggregates at micromolar and sub-micromolar concentrations. These colloids inhibit soluble proteins without specificity but with shared mechanism: adsorbing and partially denaturing them; they are the dominant artifact in early drug discovery and chemical biology. Surprisingly, the colloids are stable in cell culture, serum, and apparently even in vivo. Whereas we have previously focused on the impact of aggregates on soluble proteins in biochemical assays, we increasingly turn our attention to cell- based assays, tissue penetration and animal pharmacokinetics. The specific aims are: 1. to investigate the affect of colloids in cell culture, tissue penetration, and pharmacokinetics. We investigate the impact of aggregates on biology at increasing levels of complexity, beginning with a. Membrane-bound receptors, focusing on GPCR signaling, and continue with b. a simple, web-based tool to predict compound aggregation. These goals reflect the ongoing importance of detecting aggregation in early discovery. More ambitiously, we investigate c. Tumor penetration. Anti-neoplastic drugs like Fulvestrant aggregate not only in buffer but also in serum. Do these colloids affect the cellular activity of these drugs, and might they actually be exploited for delivery, targeting high vascular permeability of tumors? d. In vivo pharmacokinetics. Colloids of several BCS II and IV oral drugs are stable in simulated intestinal fluids. We explore their stability in the gut, and their affects on in vivo pharmacokinetics and distribution. 2. To investigate the structure and mechanism of colloidal aggregates. We continue to believe that progress and understanding will depend on fundamental physical and mechanistic studies. Key unanswered questions include: a. Thermodynamic driving forces and association kinetics. Is colloid formation driven simply by the hydrophobic effect, or are other forces at play? b. Stability and denaturation. Since colloids denature proteins, does protein stability affect the potential for colloidal inhibition? c. Mixed inhibition. Can inhibitors be wel- behaved at lower concentrations, but switch to a colloidal mechanism above some threshold? Does this lead to the common, but physically undefined, mixed inhibition mechanism? d. How might aggregators pack, and what separates them from non-aggregators? To investigate this, we will determine the small molecule crystal structures of aggregators and of close analogs that do not aggregate PUBLIC HEALTH RELEVANCE: The physical chemistry of drugs greatly influences their biology. A largely unexplored aspect of this is the propensity of many drugs and reagents to form colloids at relevant concentrations, which wholly changes their properties. Here we explore the impact of their colloids on the behavior of drugs in biological environments, including in vivo This could have a profound effect on drug discovery.